Redox regulation in O2 -tolerant [FeFe] hydrogenases: insights from two homologues

ABSTRACT O 2 -tolerance is a desirable property for [FeFe] hydrogenases, which are highly efficient H 2 -producing catalysts. While most such enzymes are highly sensitive to aerobic environments, a small number of explored representatives exhibit exceptional stability and even H 2 -producing activity under oxygenic conditions. However, the genetic signatures of the O 2 -tolerance in this class of enzymes remain largely unknown. To address this knowledge gap, we explored a close homologue of a well-characterized O 2 -tolerant [FeFe] hydrogenase from Clostridium beijerinckii ( Cb HydA1) - a hydrogenase from Terrisporobacter glycolicus ( Tg HydA1). Our investigation indeed confirms that Tg HydA1 can transition to the O 2 -stable H inact state, a hallmark of O 2 tolerance. The surprising outcome is that despite the high amino acid similarity, Tg HydA1 shows a substantially higher propensity to remain in the H inact state than Cb HydA1. Using protein film electrochemical experiments, we demonstrate that the root of this behavior lies in roughly tenfold slower reactivation rates than those of Cb HydA1 at any applied potential. This degree and direction of variation in reactivation kinetics have not been observed before for any other O 2 -tolerant [FeFe] hydrogenases or their variants to date, uncovering a yet-to-be-explored facet of reactivity alteration available to these enzymes. Overall, the results presented here highlight the importance of a holistic analysis of [FeFe] hydrogenase sequences in the context of their interaction with O 2 that encompasses the protein environment and properties of the auxiliary metallocofactors.